The complex movements responsible for chromosome segregation are powered by kinetochores, DNA-protein complexes that assemble on centromeric DNA. The goal of this work is to identify and characterize kinetochore proteins responsible for microtubule attachment and force generation in the budding yeast S. cerevisiae. This will be accomplished by combining molecular genetic dissection of kinetochore proteins with high-resolution optical tracking of centromeres in living cells. Patterns of chromosome movement will be determined in wild-type and mutant cells and force generating processes deduced - initially in a qualitative sense and subsequently in the context of a quantitative model for spindle mechanics. In the long term it is hoped that the roles of individual proteins and of multi-protein machines in force generation can be deduced. The following specific experiments will be performed: (1) The role of microtubule dynamics in chromosome movement will be investigated using mutations in tubulin genes. The recruitment to kinetochores of microtubule regulators will also be studied. (2) The involvement of newly identified kinetochore proteins to chromosome movement will be analyzed, as will the contributions of microtubule-based motor proteins. (3) Sophisticated computational tools will be developed to track chromosomes in living yeast with high accuracy, and to analyze patterns of movement rigorously. (4) The role of various proteins to kinetochore assembly will be determined in vivo using mutant strains, chromatin immunoprecipitation and imaging techniques. The significance of this work derives from its goal of describing in precise molecular terms the mechanism of action of the simplest of all known kinetochores. Kinetochores are poorly understood structures that play a critical role in ensuring the fidelity of chromosome transmission and in preventing the genetic instability and aneuploidy characteristic of tumor cells. The relevance of yeast kinetochores to human biology is emphasized by the extensive homology between the yeast proteins that are the focus of this study and as-yet poorly understood human kinetochore proteins.